Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog LeuT. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed mutagenesis and by trapping the radiolabeled cocaine analog [ 3 H]CFT in the transporter, either by cross-linking engineered cysteines or with an engineered Zn 2+ -binding site that was situated extracellularly to the predicted common binding pocket. Our data demonstrate the molecular basis for the competitive inhibition of dopamine transport by cocaine.Correspondence should be addressed to U.G. (E-mail: gether@sund.ku.dk). Note: Supplementary information is available on the Nature Neuroscience website. AUTHOR CONTRIBUTIONST.B. designed and performed the computational experiments, analyzed the data and wrote the manuscript draft together with C.J.L. J.K. generated mutants, carried out pharmacological analyses and contributed to the data analysis. M.L.B. and K.R. generated mutants and carried out pharmacological analyses. L.S. contributed to the computational experiments and manuscript refinement. L.G. participated in the design and performance of the computational experiments. A.H.N. contributed with ideas, benztropine analogues and provided expertise in the pharmacology and medicinal chemistry of DAT inhibitors. J.A.J. contributed with ideas and to the design of experiments and writing of the manuscript. H.W. directed the design and performance of the modeling and computational experiments, participated in data analysis and contributed to writing the manuscript. U.G. supervised the project together with C.J.L., designed experiments, analyzed data and wrote the final manuscript. C.J.L. supervised the project together with U.G., designed experiments, generated mutants, performed pharmacological experiments, analyzed data and wrote the manuscript draft together with T.B.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access Author ManuscriptNat Neurosci. Author manuscript; available in PMC 2009 July 1. Published in final edited form as:Nat Neurosci. 2008 July ; 11(7): 780-789. doi:10.1038/nn.2146. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCocaine is an alkaloid derived from the Peruvian Erythroxylon coca plant and has been used as a stimulant for centuries 1 . Today, cocaine is widely abused, especially in the western hemisphere, causing major socioeconomic burdens through increased medical expenses, lost earnings and increased crime 2 . Nonetheless, the molecular mechanisms underlying cocaine's pharmacology and abuse ...

Neurotransmitter:sodium symporter (NSS) proteins are secondary Na(+)-driven active transporters that terminate neurotransmission by substrate uptake. Despite the availability of high-resolution crystal structures of a bacterial homolog of NSSs-Leucine Transporter (LeuT)-and extensive computational and experimental structure-function studies, unanswered questions remain regarding the transport mechanisms. We used microsecond atomistic molecular-dynamics (MD) simulations and free-energy computations to reveal ion-controlled conformational dynamics of LeuT in relation to binding affinity and selectivity of the more extracellularly positioned Na(+) binding site (Na1 site). In the course of MD simulations starting from the occluded state with bound Na(+), but in the absence of substrate, we find a spontaneous transition of the extracellular vestibule of LeuT into an outward-open conformation. The outward opening is enhanced by the absence of Na1 and modulated by the protonation state of the Na1-associated Glu-290. Consistently, the Na(+) affinity for the Na1 site is inversely correlated with the extent of outward-open character and is lower than in the occluded state with bound substrate; however, the Na1 site retains its selectivity for Na(+) over K(+) in such conformational transitions. To the best of our knowledge, our findings shed new light on the Na(+)-driven transport cycle and on the symmetry in structural rearrangements for outward- and inward-open transitions.

Importance Animal studies have suggested that prenatal cocaine exposure (PCE) deleteriously influences the developing nervous system, in part attributable to its site of action in blocking the function of monoamine reuptake transporters, increasing synaptic levels of serotonin and dopamine. Objective To examine the brain morphologic features and associated impulsive behaviors in adolescents following prenatal exposure to cocaine and/or tobacco. Design Magnetic resonance imaging data and behavioral measures were collected from adolescents followed up longitudinally in the Maternal Lifestyle Study. Setting A hospital-based research center. Participants A total of 40 adolescent participants aged 13 to 15 years were recruited, 20 without PCE and 20 with PCE; a subset of each group additionally had tobacco exposure. Participants were selected and matched based on head circumference at birth, gestational age, maternal alcohol use, age, sex, race/ethnicity, IQ, family poverty, and socioeconomic status. Main Outcome Measures Subcortical volumetric measures of the thalamus, caudate, putamen, pallidum, hippocampus, amygdala, and nucleus accumbens; cortical thickness measures of the dorsolateral prefrontal cortex and ventral medial prefrontal cortex; and impulsivity assessed by Conners' Continuous Performance Test and the Sensation Seeking Scale for Children. Results After controlling for covariates, cortical thickness of the right dorsolateral prefrontal cortex was significantly thinner in adolescents following PCE (P=.03), whereas the pallidum volume was smaller in adolescents following prenatal tobacco exposure (P=.03). Impulsivity was correlated with thalamic volume following either PCE (P=.05) or prenatal tobacco exposure (P=.04). Conclusions and Relevance Prenatal cocaine or tobacco exposure can differentially affect structural brain maturation during adolescence and underlie enhanced susceptibility to impulsivity. Additional studies with larger sample sizes are warranted.

Objective: To examine the independent contributions of prenatal methamphetamine exposure (PME) and prenatal tobacco exposure (PTE) on brain morphology among a sample of nonalcohol-exposed 3- to 5-year-old children followed prospectively since birth. Study Design: The sample included 20 children with PME (19 with PTE) and 15 comparison children (7 with PTE), matched on race, birth weight, maternal education and type of insurance. Subcortical and cortical volumes and cortical thickness measures were derived through an automated segmentation procedure from T1-weighted structural magnetic resonance images obtained on unsedated children. Attention was assessed using the computerized Conners’ Kiddie Continuous Performance Test Version 5 (K-CPT™ V.5). PME effects on subcortical and cortical brain volumes and cortical thickness were tested by general linear model with type III sum of squares, adjusting for PTE, prenatal marijuana exposure, age at time of scan, gender, handedness, pulse sequence and total intracranial volume (for volumetric outcomes). A similar analysis was done for PTE effects on subcortical and cortical brain volumes and thickness, adjusting for PME and the above covariates. Results: Children with PME had significantly reduced caudate nucleus volumes and cortical thickness increases in perisylvian and orbital-frontal cortices. In contrast, children with PTE showed cortical thinning in perisylvian and lateral occipital cortices and volumetric increases in frontal regions and decreases in anterior cingulate. PME was positively related and caudate volume was inversely related to K-CPT reaction time by inter-stimulus interval, a measure of the ability to adjust to changing task demands, suggesting that children with PME may have subtle attentional deficits mediated by caudate volume reductions. Conclusions: Our results suggest that PME and PTE may have distinct differential cortical effects on the developing central nervous system. Additionally, PME may be associated with subtle deficits in attention mediated by caudate volume reductions.

␤-Amyloid (A␤) accumulation is a pathological hallmark of Alzheimer's disease (AD), and during the past decade there has been increasing evidence for a critical role for the accumulation of A␤ within neurons in AD. Many studies have shown early intraneuronal A␤42 accumulation in AD, Down syndrome, and familial Alzheimer's disease (FAD) mutant transgenic rodents.1-18 Importantly, in a triple transgenic mouse, intraneuronal A␤ accumulation was the earliest pathological correlate of abnormalities in long-term potentiation and behavior. 19,20 Thioflavin S (ThS)-positive intraneuronal A␤ fibrils have been described in both APP SL PS1KI and 5XFAD transgenic mice. 21,22 Moreover, neuron loss correlated with the prior appearance of ThS-positive intraneuronal A␤ fibrils in transgenic mice.22 By immunoelectron microscopy, A␤42 normally localizes to endosomal vesicles. A␤42 increases with aging, particularly at the outer membranes of multivesicular bodies and smaller endosomal vesicles, and particularly in distal processes and synaptic compartments, where such A␤ accumulation can be directly associated with ultrastructural pathology. 6,23 The development of new imaging software allows for higher resolution image analysis of immunofluorescence confocal microscopy. Volume-rendering techniques display two-dimensional stacks of images as 3D volumetric images. High-resolution three-dimensional (HR-3D) volumetric image analysis provides a more complete 3D view of A␤ pathology, leading to new insights into neurite and synapse disruption. In the present study, we provide HR-3D evidence for intracellular A␤ accumulation and fibrillization within synapses, and distal and proximal processes of neurons in CA1 hippocampus. These images demonstrate that before the onset of plaque pathology, intraneuronal A␤ accumulation and fibrillization can de-

The accurate modeling of protein-ligand interactions, like any prediction of macromolecular structure, requires an energy function of sufficient detail to account for all relevant interactions and a conformational search method that can reliably find the energetically favorable conformations of a heterogeneous system. Both of these prerequisites represent daunting challenges. Consequently, the routine docking of small molecules or peptides to proteins in their correct binding modes, and the reliable ranking of binding affinities remain unsolved problems. Nonetheless, computational techniques are continually evolving so as to broaden the range of feasible applications, and the accuracy of predictions and theoretical approaches can often be of great help in guiding and interpreting experiments. We discuss the energetics of protein-ligand systems and survey conformational searching techniques. We illustrate how molecular modeling of a protein-ligand complex sheds light on the observed resistance of a mutant dihydrofolate reductase to the antibiotic trimethoprim. In another example, we show that relaxation of side chains in different crystal structures of the same complex, benzamidine bound to trypsin, is needed to draw sensible conclusions from the calculations. The results of these relatively simple conformational searches underscore the importance of incorporating protein flexibility in simulations of protein-ligand interactions, even in the context of relatively rigid binding pockets.

Background: β-Amyloid (Aβ) plaques are a pathological hallmark of Alzheimer’s disease (AD) and multiple lines of evidence have linked Aβ with AD. However, synapse loss is known as the best pathological correlate of cognitive impairment in AD, and intraneuronal Aβ accumulation has been shown to precede plaque pathology. The progression of Aβ accumulation to synapse loss and plaque formation remains incomplete. The objective is to investigate the progression of intraneuronal Aβ accumulation in the brain. Methods: To visualize and analyze the development of Aβ pathology we perform immunohistochemistry and immunofluorescence microscopy using antibodies against different Aβ conformations, synaptic proteins and structural neuronal proteins in brain tissue of AD transgenic mouse models. Results: Our results show the intraneuronal onset of Aβ42 accumulation in AD mouse brains with aging. We observe an inverse correlation of Aβ and amyloid fibrils with structural proteins within neurites. Images reveal aggregated amyloid within selective pyramidal neurons, neurites and synapses in AD transgenic mice as plaques arise. Conclusion: The data support that Aβ42 accumulation and aggregation begin within AD-vulnerable neurons in the brain. Progressive intraneuronal Aβ42 aggregation disrupts the normal cytoarchitecture of neurites.

The conformational profiles for the endogenous peptide Opiorphin and a set of seven analogues exhibiting different inhibitory activities toward human aminopeptidase N (hAPN) and human neprilysin (hNEP) were independently computed to deduce a bioactive conformation that Opiorphin may adopt when binding these two enzymes. The conformational space was thoroughly sampled using an iterative simulated annealing protocol, and a library of low-energy conformers was generated for each peptide. Bioactive Opiorphin conformations fitting our experimental structure-activity relationship data were identified for hAPN and hNEP using computational pairwise comparisons between each of the unique low-energy conformations of Opiorphin and its analogues. The obtained results provide a structural explanation for the dual hAPN and hNEP inhibitory activity of Opiorphin and show that the inborn flexibility of Opiorphin is essential for its analgesic activity.